Anti-mitotic drugs, such as taxanes and vinca alkaloids that are directed at inhibiting the dynamic function of microtubules (MTs) have proved to be effective in the treatment of cancer. However, the use of these conventional MT-targeting agents is severely constrained by their dose-limiting toxicities due to the wide-spread functions of MTs in mediating both mitotic and non-mitotic cellular processes in many cell types, notably intestinal epithelia cells and lymphocytes. Therefore, developing a new class of anti-cancer therapeutics by targeting mitosis-specific proteins critical for cancer cell proliferation is an important line of investigation. Polo-like kinase 1 (Plk1) is one of the most attractive targets for anti-cancer therapy. Efforts to generate Plk1-specific inhibitors by targeting the catalytic activity of Plk1 have proven to be difficult due to similarities with the catalytic domains of other structurally related kinases. Here, we propose to develop a new class of mono-specific Plk1 inhibitors by employing a novel approach of targeting the non-catalytic, but functionally essential, PBD of Plk1. To this end, we have carried out a high throughput screen in collaboration with NCATS, Bethesda, MD. From this screen, we have identified 3,000 compounds from a primary screen, which were narrowed down to the final 2 compounds (1S and B7) through secondary medium throughput and tertiary cell-based assays. Through systematic molecular modeling/docking of these parent compounds, we obtained six significantly improved compounds, which showed Plk1 PBD inhibition activity at levels similar to that of one of the previously characterized PBD-binding ligands, PLHSpT (Kd = 450 nM), in an in vitro ELISA. Since the original HTS leads may belong to a class known as pan-assay interference compounds, or PAINS, we will further incorporate many drug-like characteristics during the hit-to-lead optimization in order to obtain a high-impact chemical probe that ultimately exhibits the desired efficacy against the Plk1 PBD in proof-of-concept mouse tumor models. As the first step of drug discovery, we generated the co-crystal structure of Plk1 PBD with one of the lead compounds. This structural model will be used to carry out structure-activity relationship studies and further optimization of the compounds. As a result of this effort, we obtained several initial hits, which have been subjected to several rounds of in silico derivatization and ELISA-based Plk1 PBD inhibition analysis. These efforts led to the discovery of seven compounds (NC21 to NC27) with distinct chemotypes. In vitro FP and ELISA assays with purified compounds confirmed their anti-PBD specificity with Kd values in the range of 100-200 nM. For the past two years, we have carried out hit-to-lead optimizations (in collaboration with Dr. Ken Jacobson, NIDDK, NIH) and developed 20 inhibitors that appear to potently inhibit Plk1 PBD inhibitors in vitro assays. However, the efficacy and bioavailability of these inhibitors exhibit less than expected. Now, the effort is being focused on improving both the potency and permeability. Currently, we are wrapping up our structure-activity-relationship studies to submit this early data for publication.